The long-term objective of this proposal is to develop an innovative structural, biophysical, and functional biochemical framework to advance our understanding of HIV splicing mechanisms. The HIV genome is synthesized as a single polycistronic RNA transcript that undergoes regulated splicing to ensure mRNA homeostasis. Aberrations in the viral splicing machinery impair HIV replication fidelity and in some cases render the virus less infectious. Thus, HIV splicing pathways represent novel targets for therapeutic intervention. Splicing decisions are determined by the dynamic assembly/disassembly of trans host factors with cis viral RNA control elements; however, the underlying molecular events that determine whether a splice site will be activated or repressed remain elusive. Knowledge of the physical and functional determinants of how one highly conserved splice site gets differentially regulated by two trans host factors offers the opportunit to define a mechanism. Hence, the immediate goal of this proposal is to identify the molecular principles that HIV uses to co-opt the mutually antagonistic factors, hnRNP A1 (A1) and ASF, in order to regulate its 3' splice site A7 (ssA7). Furthermore, A1 and ASF regulate other conserved HIV splice sites via poorly characterized mechanisms; thus, the work proposed here is likely to have broad implications on our understanding of HIV splicing in general and may lead to new approaches to thwart HIV replication. The specific aims of this project are to: (1) Determine the contribution of structure to target site selection by A1 and ASF, (2) Elucidate the bio-thermodynamic underpinnings of mutual antagonism between A1 and ASF, and (3) Investigate the molecular origins of cooperativity in ssA7 regulation. PUBLIC HEALTH RELEVANCE: Narrative: HIV, the etiological agent of AIDS, continues to be a major health threat infecting more than 30 million people globally. A better understanding of the fundamental principles of HIV replication may serve as an underpinning to identify novel therapeutic targets and mechanisms of intervention. Towards that end, this proposal seeks to elucidate molecular mechanisms of HIV regulated splicing.